The diketopiperazines (DKPs)-containing natural products represent a large class of secondary metabolites mainly produced by bacteria, fungi, marine invertebrates and higher organisms. DKPs were biosynthesized by condensation of two amino acids including tryptophan, proline, histidine and phenylalanine.1, 2, 3 Interest in DKPs is due to their activities in various pharmacological assays ranging from antibacterial, antifungal, antiviral to immunosuppressive.4, 5, 6, 7 In recent studies, the nocazines are a newly defined family of antibacterial and cytotoxic cyclic dipeptides produced by Nocardiopsis dassonvillei and N. alba.8, 9 Our investigation with a deep-sea sediment strain, YIM M13066, identified as a Nocardiopsis sp., was found to produce six DKPs, including two new DKPs, nocazines F (1) and G (2), and four known DKPs (3–6). In this study, the fermentation, isolation, and structural elucidation and bioactivity of compounds 1–6 (Figure 1) from Nocardiopsis sp. YIM M13066 were described.

Figure 1
figure 1

The chemical structures of compounds 1–6.

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The strain YIM M13066 was first cultured in Petri dishes with ca. 20 ml MP agar medium for 7 days to obtain seed cultures. The seeds were inoculated on MP agar media (20 l) by streaking plate method and cultured for 10 days at 28 °C. The fermented culture was extracted with EtOAc–MeOH (85:15, volume per volume, 20 l) at room temperature to obtain a crude extract. The crude extract was partitioned with equal volume EtOAc (0.5 l) and H2O. The EtOAc portion was concentrated under vacuum to afford the EtOAc extract (7.8 g). The extract was subjected to column chromatography over Sephadex LH-20 (25–100 μm; GE Healthcare, Sweden; column dimensions: 200 × 20 mm) eluted with MeOH and pooled on the basis of TLC detection to obtain Fr.1–10. Fr.7 (1.1 g) was separated by MPLC (310 × 2.6 mm) over RP-18 silica gel (80 g) to afford Fr.7a–7h. Compounds 5 (30.0 mg) and 6 (40 mg) were obtained by crystallization from Fr.7d and Fr.7e, respectively. Fr.7f was purified by HPLC (SunFire Prep C18 OBD, 19 × 150 mm, 15 ml min−1, UV 365 nm) eluted with 70% acetonitrile to afford 4 (tR 11.7 min, 6.8 mg) and 3 (tR 13.5 min, 7.1 mg). Fr.10 (108.0 mg) was separated by MPLC over RP-18 silica gel (80 g) to afford Fr.10a–10j. Fr.10e was purified by HPLC (SunFire Prep C18 OBD, 19 × 150 mm, 15 ml min−1, UV 365 nm) eluted with 40% acetonitrile to yield 1 (tR 7.1 min, 29.2 mg) and 2 (tR 9.2 min, 20.1 mg), respectively.

Nocazine F (1) was obtained as a yellow amorphous powder with the molecular formula C20H18N2O4 according to its HRESI-MS at m/z=351.1297 [M+H]+ (calcd 351.1339). Its 1H and 13C NMR (in DMSO-d6, Table 1) corresponded to 1 NH group (δ 9.86 (s)), 2 OCH3 (δH/δC 3.98/54.8 and 3.79/55.7), 10 olefinic CH, 7 quaternary C-atoms and 1 amidocarbonyl (δ 160.2). The presence of two 1,4-disubstituted benzene rings was determined by two sets of coupled 1H signals at δ 8.02 (d, J=8.7 Hz), 6.83 (d, J=8.7 Hz) and δ 7.50 (d, J=8.7 Hz), 6.99 (d, J=8.8 Hz). HMBC correlations from 18-OH (δ 9.91) to C-17/19 (δ 115.9) and C-18 (δ 158.8), and from 11-OCH3 (δ 3.79) to C-11 (δ 159.5) revealed that the two 1,4-disubstituted benzene rings were connected with hydroxyl and methoxyl, respectively. HMBC correlations from 5-OCH3 (δ 3.98) to C-5 (δ 154.6), from NH-1 (δ 9.86) to C-5 and C-3, from H-7 (δ 6.46) to C-5 and C-9/13 (δ 131.2), and from H-14 (δ 7.03) to C-2 (δ 160.2) and C-16/20 (δ 133.8) indicated a specific DKP unit formed from a p-hydroxyl-Phe (Tyr) and a p-MeO-Phe moieties, similar to nocazine A.8 The Z configurations of both Δ6 and Δ3(14) double bonds were determined by the NOE correlations between 5-OCH3 (δ 3.98) and H-20 (δ 8.02), NH-1 (δ 9.86), and H-13 (δ 7.50) (Figure 2). Finally, the structure of compound 1 was determined as 3-((Z)-4-hydroxybenzylidene)-5-methoxy-6-((Z)-4-methoxybenzylidene)-3,6-dihydropyrazin-2(1H)-1.

Table 1 The 1H and 13C NMR data for 1 and 2 (δ in p.p.m., J in Hz)
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Figure 2
figure 2

Selected HMBC, COSY and NOESY correlations of compound 1.

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Nocazine G (2) was obtained as a yellow amorphous powder. Its molecular formula was determined as C21H20N2O4 according to its HRESI-MS at m/z=365.1503 [M+H]+ (calcd 365.1496). The NMR spectra of 2 were very similar to those of nocazine A.8 The difference is that the 18-OCH3 in nocazine A was replaced by 18-OH in 2. The Z configurations of both Δ6 and Δ3(14) double bonds were determined by the NOE correlations between 5-OCH3 (δ 4.04) and H-20 (δ 8.05), 1-N-CH3 (δ 3.00), and H-13 (δ 7.19). Furthermore, the Z configurations of the two double bonds in 2 also can be deduced from the similarity of the chemical shifts of H-7 (δ 6.80) and H-14 (δ 7.22) to those of nocazine A and B8 (in CDCl3). Therefore, compound 2 was determined as 3-((Z)-4-hydroxybenzylidene)-5-methoxy-6-((Z)-4-methoxybenzylidene)-1-methyl-3,6-dihydropyrazin-2(1H)-1.

Compounds 3–6 were found to be nocazine A (3),8 nocazine B (4),8 XR 330 (5)9, 10 and (3Z,6Z)-3-(4-methoxybenzylidene)-6-(2-methylpropylidene)-piperazine-2,5-dione (6),9, 11 respectively, by comparing their NMR and MS data with those previously reported.

The new compounds (1 and 2) were evaluated for in vitro cytotoxic activities against human cancer cell lines H1299, HeLa, HL7702, MCF-7, PC3 and U251 using the sulforhodamine B method,12 and VP-16 was used as a positive control, and for antibacterial activities against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6051, Pseudomonas aeruginosa PA01 and Salmonella enterica serovar Typhimurium UK-1 χ8956. Compounds 1 and 2 showed broad spectrum and excellent cytotoxicity to all tested cell lines (Table 2). As for antimicrobial activity, only 2 showed moderate activities against B. subtilis ATCC 6051 with an MIC of 25.8 μm. Meanwhile, compounds 1–6 were further assayed for their activities of inhibiting the type three secretion system (T3SS) of S. enterica serovar Typhimurium UK-1 χ8956 using cytosporone B as the positive control, as described.13 Compounds 1 –6 exhibited inhibitory activity against the secretion of SPI-1 effector SipA/B/C/D and no effects on FliC at the concentration of 100 μm (Figure 3). Among them, compound 1 exhibited a strong inhibition on the secretion of the SPI-1 effector SipC in a dose-dependent manner (Figure 4).

Table 2 IC50 values (μm) of compounds 1 and 2 tested against human tumor cell lines
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Figure 3
figure 3

The effects of compounds 1–6 on secretion of SPI-1 effector proteins. Csn-B, cytosporone B; FliC, flagellar filament protein; SipA/B/C/D, SPI-1 effector proteins. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Figure 4
figure 4

The effect of compound 1 on secretion of SPI-1 effector SipC in a dose-dependent manner. Csn-B, cytosporone B; FliC, flagellar filament protein; SipA/B/C/D, SPI-1 effector proteins. A full color version of this figure is available at The Journal of Antibiotics journal online.

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In summary, the chemical study on strain YIM M13066 led to the isolation and characterization of six DKPs (1–6). Compared with nocazine A-D,8, 9 XR330 and 334,10 nocazines F (1) and G (2) showed a broad spectrum of cytotoxicity against the human tumor cell lines, which suggested that the O-methyl group at C-5, 18-OH group and the Δ6(7) double bond may be the key active groups of nocazines. Besides, nocazine F exhibited a strong inhibition on the T3SS of S. enterica serovar Typhimurium, which could be a promising candidate for a novel type of antibiotics.